Thermostable lipase isolated from Pseudomonas solanacearum

ABSTRACT

A thermostable lipase has been isolated from Pseudomonas solanacearum SD709 (FERM BP-5358) which has a mass of 32 kD by SDS-PAGE, enzymatic activity in a pH range of about 4-12, a pH optimum of 6.5-9.5 and a temperature optimum of 80°-90° C.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/JP96/00454 filed Feb. 27, 1996 and claims priority under 35 U.S.C.119 of Japanese application 7-45803 filed Mar. 6, 1995, the contents ofwhich are fully incorporated herein by reference.

TECHNICAL FIELD

This invention relates to a novel lipase, a production method thereofand a microorganism producing the same, and more specifically, to anovel thermostable lipase showing high activity at high temperaturewhich is produced by a bacterium belonging to Pseudomonas solanacearum,a microorganism producing the lipase and production method thereof.

BACKGROUND ART

A lipase is a general term of an enzyme which hydrolyze triglyceride,and has formerly been produced by extraction of internal organs ofanimals or by using microorganisms.

The lipase is extensively used as an enzyme for food processing toflavor dairy products, medicines as digestives, diagnosis of a bloodlipid assay, industry in hydrolysis and improvement of fats and oils,and the like. The lipase is required to have various characteristics foreach use, and a thermostable lipase is applied to a wide variety offields and requested to be variously used.

In food processing, from a food-hygienic point of view, enzyme reactionat high temperature which is in low danger of bacterial contaminationhas been desired. In decomposition of fats and oils, application of thelipase has been considered. Hydrolysis of fats and oils is a process forproducing fatty acid and glycerol which are materials of petrochemicalproducts such as detergents, cosmetics and surface-active agents. Atpresent the Colgate-emery method is mainly carried out which brings fatsand oils into contact with steam at 250° to 260° C. and at 50 to 55atmospheres, however, the method needs heavy facilities and is notappropriate for small-to-medium-scale production of soap, etc.Therefore, decomposition of fats and oils by a lipase has beeninvestigated.

However, stearic acid (melting point: 67° to 70° C.) and palmitic acid(melting point: 63° to 640° C.) which constitute a variety of fats andoils are solid at ordinary temperature. For that reason, the reactionfats and oils must be reacted at the temperature above their meltingpoints. Therefore, the lipase used for decomposition of fats and oilsmust have a high thermostability and a high reactivity at a hightemperature.

Furthermore, in recent years, the lipase has been used for a solution ofpitch troubles in paper-manufacturing industry (Japanese Examined PatentPublication No.

Hei. 4-29794). The optimum temperature of the enzyme reactionconventionally adopted for a solution of pitch troubles is from 35° to55° C. Since the conventional enzyme is inactivated above 70° C., thetemperature of the enzyme reaction in the paper-manufacturing process islimited and the temperature must be controlled throughout thepaper-manufacturing process in which the lipase is used. Also, theenzyme is not thermostable, which is one of the causes to inhibit theuse of the enzyme for a solution of pitch troubles at the grindertreatment part.

At present, most of the well-known enzymes on the market used as athermostable lipase are not adequate for the stability against heat andnot practical. For that reason, a Japanese Unexamined Patent PublicationNo. Sho. 62-79782 proposed a thermostable lipase. However, the optimumtemperature of the enzyme is from 60° to 70° C., and its thermostabilityis not sufficient, since the residual activity after treatment at 70° C.for 15 minutes is below 10%. Moreover, because the enzyme hardly acts ontriacetin and tributyrin, its use is restricted to food-processing. Athermostable lipase derived from Rhizopus has been disclosed (JapaneseUnexamined Patent Publication Number Sho. 59-156282), but the optimumtemperature of the enzyme is 60° C. and the reactivity at a hightemperature is not sufficient. Furthermore there are the reports of alipase produced by Pseudomonas mephitica var.iipolytica which has theoptimum temperature of 70° C. and is not inactivated by heat treatmentat 60° C. for 14 hours (Japanese Examined patent Publication No. Sho.50-25553) and that produced by Pseudomonas fraji which has an optimumtemperature of from 75° to 80° C. and maintains 95% of the activityafter heat treatment at 70° C. for 20 minutes (Agric.Biol.Chem.41,1353-1358(1977)). However, the optimum temperature of these enzymesis not beyond 80° C., and also concerning thermostability, the activitythereof is reduced to some degree by treatment at 80° C. for 1 hour, andtherefore they are not sufficiently satisfactory for thermostability.

PURPOSE OF THE INVENTION

As mentioned above, the well-known lipase is not sufficient forthermostability and reactivity at a high temperature, so that theopportunity to use the lipase practically has been little in the fieldssuch as food-processing, industry and a paper-manufacturing process, andthere have been problems of the temperature condition being restrictedin the process using lipase.

Consequently, an object of the present invention is to provide a lipasehaving a high thermostability. Also, another object is to provide aproduction method thereof, the lipase having an excellentthermostability, and a bacterium producing the thermostable lipase.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the relationship between the reactive pH andthe relative activity of the lipase produced by SD709.

FIG. 2 is a graph showing the residual activity of the lipase producedby SD709 after it was maintained at varying pH at 37° C. for 1 hour.

FIG. 3 is a graph showing the relationship between the reactiontemperature and the relative activity of the lipase produced by SD709.

FIG. 4 is a graph showing the residual activity of the lipase producedby SD709 after it was treated at a variety of temperatures at pH 7 for 1hour.

FIG. 5 is a graph showing the relationship between the reactingtemperature in the presence and absence of EDTA and the relativeactivity of the lipase produced by SD709.

DISCLOSURE OF THE INVENTION

The present inventors isolated, cultivated and screened a large numberof microorganisms to obtain a lipase having a high thermostability and ahigh reactivity at a high temperature, and found out that the strainrepresented by Pseudomonas solanacearum SD709 (FERM P-14786), which hadbeen isolated from the soil in Chiba prefecture in Japan, produces anovel thermostable lipase. Until now the lipase produced by numbers ofstrains has been known, but the thermostable lipase derived fromPseudomonas solanacearum has not yet been known. The inventors confirmedthat the novel thermostable lipase is extremely effective for hydrolysisof lipids at a high temperature to achieve the invention. That is, theinvention provides a lipase given below, a production method thereof anda microorganism producing the same.

1) A lipase having the active temperature of from 30° to 100° C. and theoptimum temperature of from 85° to 100° C. determined using trioleinemulsion as the substrate in the range of from 30° to 100° C.,preferably, is a lipase having the properties below.

(1) Active pH and Optimum pH

The active pH is from 4 to 12 and the optimum pH is from 6.5 to 9.5,which are determined using triolein emulsion as the substrate in the pHrange of from 4 to 12.

(2) Molecular Weight

The molecular weight determined by SDS-polyacrylamide gelelectrophoresis is 32,000±2,000.

2) A lipase having the active temperature of from 30° to 100° C. and theoptimum temperature of from 80° to 90° C. determined using trioleinemulsion as the substrate in the range of from 30° to 100° C. in thepresence of 5 mM EDTA, preferably, is a lipase having the propertiesbelow.

(1) Active pH and Optimum pH

The active pH is from 4 to 12 and the optimum pH is from 6.5 to 9.5which are determined using triolein emulsion as the substrate in the pHrange of from 4 to 12.

(2) Molecular Weight

The molecular weight determined by SDS-polyacrylamide gelelectrophoresis is 32,000±2,000.

3) A lipase described in the 1) or 2) which is obtained from the culturemedium of the bacterium which belongs to Pseudomonas, preferably, saidbacterium belonging to Pseudomonas is Pseudomonas solanacearum.

4) A lipase described in 1) or 2) which is obtained from the culturemedium of Pseudomonas solanacearum SD709 (FERM P-14786).

5) A bacterium described in 1) or 2) belonging to Pseudomonas whichproduces a lipase, preferably, Pseudomonas solanacearum, morepreferably, Pseudomonas solanacearum SD709 (FERM P-14786), or amycologically equivalent or their mutants.

6) A production method of a lipase which comprises cultivating thebacterium described in the 5) and obtaining the lipase described in 1)or 2) from the culture medium.

Producing Strain!

The microorganism used for the production of the lipase according to thepresent invention is not especially limited so long as the bacterium canproduce a lipase having the properties described below. Such a bacteriumcan be selected from preserved strains or microorganisms newly isolatedfrom the natural world, a preferable bacterium is one belonging toPseudomonas, and a more preferable one is belonging to Pseudomonassolanacearum. Further preferably, it is Pseudomonas solanacearum SD709and its mycologically equivalents. In the present application, amycologically equivalent mean strains having the almost same mycologicalproperties. The equivalents naturally include natural or artificialmutant so long as they have the properties described below.

An example of the strains producing the novel lipase of the invention isSD709 which was isolated from the soil in Chiba prefecture in Japan bythe present inventors.

The SD709 has the properties below.

    ______________________________________                                        (1)  Morphology:          Rod                                                 (2)  Gram Stain:          Negative                                            (3)  Spore:               (-)                                                 (4)  Motility:            (+)                                                 (5)  Flagella:            Polar multiple flagella                             (6)  Oxidase:             Positive                                            (7)  Catalase:            Positive                                            (8)  OF test:             O                                                   (9)  Production of Fluorescent Pigment:                                                                 (-)                                                 (10) Production of Water-Soluble Pigment:                                                               (+)                                                 (11) Cleavage of Protocatechuic Acid:                                                                   Ortho                                               (12) Arginine Dihydrase   Negative                                            (13) Growth at 41° C.:                                                                           Impossible                                          (14) Denitrification:     Positive                                            (15) Gelatin Liquefaction:                                                                              Positive                                            (16) Starch Decomposition:                                                                              Negative                                            (17) PHB Accumulation:    (+)                                                 (18) Assimilation                                                                  Glucose:             +                                                        D-Xylose:            -                                                        D-Ribose:            +                                                        L-Rhamnose:          -                                                        Levulinate:          +                                                        Citraconate:         +                                                        Mesaconate:          -                                                        Adonitol:            -                                                        2,3-Butylene Glycol: -                                                        m-Hydroxybenzoic Acid:                                                                             -                                                        Tryptamine:          -                                                        Sucrose:             +                                                        Caprylate:           +                                                        L-(+)-Tartrate:      +                                                   (19) Quinones:            Q-8                                                 ______________________________________                                    

The classificatory properties of the bacterium having the mycologicalproperties described above were compared with other strains by referenceto Bergey's Manual of Systematic Bacteriology (1984) and, as a result,the strain was identified as Pseudomonas solanacearum. The strain wasdeposited in National Institute of Science and Human-Technology Agencyof Industrial Science and Technology (1-1-3, Higashi, Tsukuba, Ibaragi,Japan) on Feb. 23, 1995 as P-14786 and has been transferred to theinternational deposit in accordance with the Budapest treaty since Dec.28, 1995 as Pseudomonas solanacearum (Pseudomonas sp.) SD709 (FERMBP-5358).

A mutant producing a lipase which has the properties described below canbe obtained by spontaneous or induced mutation from the strain above asthe parent strain, and therefore the mutant can be used as the lipaseproducing bacterium. A conventional method to prepare the mutant is, forexample, a method which comprises carrying out mutagenesis with anartificial means such as ultraviolet irradiation, treatment withN-methyl-N'-nitro-N-nitrosoguanidine (NTG), etc. for the parent strain,spreading that on nutrient agar containing oil such as olive oil, etc.,selecting colonies in which the clear zones formed around the coloniesare larger, cultivating them with a lipase production medium, andselecting the strain of excellent productivity.

Production!

The lipase according to the invention is mainly obtained in the culturemedium by cultivating the lipase producing bacterium. For a nutrient ofthe medium, usual ones are widely applicable. As carbon sources, anassimilative carbon compound or substance containing it can be used suchas glucose, fats and oils, corn steep liqueur, Tween surface-activeagents, etc. As nitrogen sources, a assimilative nitrogen compound orsubstances containing it can be used such as ammonium salt, nitrate, soybean powder, meat extract, corn steep liqueur, pharmamedia. As inorganicsalt, salt such as phosphate, magnesium salt, calcium salt, manganesesalt is appropriately used.

The cultivation condition varies with the compositions of the culturemedium, but the proper condition for production of the desired lipase ischosen. Usually, the cultivation temperature is from 10° to 35° C.,preferably from 20° to 30° C., and the cultivation period isapproximately from 8 to 100 hours, the cultivation is terminated whenthe production of the lipase reaches maximum. The preferable pH of theculture medium for production of the lipase is from 7 to 10. By means ofsuch cultivation, the desired lipase is mainly produced extracellularly(in the culture medium).

Separation and Purification Method!

To recover the lipase from the culture medium obtained in the abovemanner, a conventional method can be carried out by separation andpurification to recover the lipase.

That is, the supernatant or filtrate obtained by separating thebacterial cells and solid medium from the culture medium is separated bywell-known appropriate method such as filtration, centrifugation, andthe separated solution, which may be concentrated or not, is treated byone or more of the separation or purification means such as salting-outin which the enzyme is precipitated by addition of soluble salt, organicsolvent precipitation in which the enzyme or impurities is precipitatedby addition of a hydrophilic organic solvent, adsorption/ desorptionmethod using ion-exchange resin, gel filtration, spray drying with orwithout addition of stabilization, freeze drying, to give the lipase.

Method of Enzyme Activity Assay!

The assay of the lipase activity was carried out by the assay usingtriolein-poly(vinyl alcohol) (PVA) emulsion as the substrate. Theembodiment of the activity assay is described below.

The mixed solution consisting of 0.1 ml of the enzyme solution, 0.4 mlof 200 mM tris-buffer (pH 9.0) and 0.5 ml of triolein emulsion washeated in a test tube with an airtight stopper at 37° C. for 10 minutesto react, and the reaction was stopped by the addition of 0.2 ml of 1Nhydrochloric acid as the reaction stopper. The triolein emulsion usedhere was prepared by adding 2.5 g of triolein to 10 ml of 2% aqueoussolution of PVA (Poval PVA117 (trade name, Kuraray Co., Ltd.):PovalPVA205 (trade name, Kuraray Co., Ltd.)=9:1) and homogenizing it with icecooling at 18000 rpm for 10 minutes. After the reaction was stopped, 2ml of n-hexane, 2 ml of isopropyl alcohol and 1 ml of distilled waterwere added, vigorously stirred and allowed to stand, then, the oleicacid in a sample from the hexane layer was determined by TLC-FID method(Minagawa et al., Lipids, 18,732,1983). Unit (U) of activity was definedas the quantity of the enzyme generating 1 micro mole of oleic acid for1 minute.

Enzyme Property!

As an example of the lipase of the present invention, the properties ofthe lipase produced by Pseudomonas solanacearum SD709 mentioned aboveare described below.

(1) Action

It acts on glyceride and hydrolyzes its ester.

(2) Substrate Specificity

It widely hydrolyzes a variety of glyceride, ester, etc. The relativeactivity was determined by the enzyme activity assay described aboveusing each glyceride-PVA emulsion as the glyceride substrate. Therelative activities were 180 on tributyrin, 75 on olive oil, 90 on soybean oil and 74 on cotton oil comparing with 100 in the decompositionpower on triolein.

The decomposition ability on ester was determined by colorimetry withp-nitrophenol (OD405) generated from hydrolysis at pH 8.0 and 30° C.using p-nitrophenyl fatty acid ester as the substrate.

The relative activities were 170 on p-nitrophenyllaurate (pNPL) and 60on p-nitorphenylvalerate (pNPV) comparing with 100 in the decompositionpower on p-nitrophenylpalmitate (pNPP).

(3) Active pH and Optimum pH

They were determined by the enzyme activity assay described above usingtriolein emulsion as the substrate. The pH in the reaction wasdetermined in various pH in the range of from 4 to 12. The mixed bufferconsisted of 100 mM e-aminocapronic acid, 100 mMbis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (bistris) and 100mMN-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), andthe pH of it was adjusted with hydrochloric acid or sodium hydroxidebefore use. The relationship between the reactive pH and the relativeactivity is shown in FIG. 1. The active pH determined in the range offrom 4 to 12 was from 4 to 12, and the optimum pH was from 6.5 to 9.5.

(4) pH Stability

The residual activity after treatment in varying pH in the range of from4 to 12 at 37° C. for 1 hour was determined by the enzyme activity assaydescribed above. The relationship between the treatment pH and theresidual activity was shown in FIG. 2, and the residual activity in thepH range of from 4 to 11 is not less than 50%. The buffer used for thetreatment comprised the followings; pH 4-5: acetic acid/sodium acetate,pH 6-7:phosphoric acid, pH 8-9:tris/hydrochloric acid, pH10-12:glycine/sodium hydroxide.

(5) Active Temperature and Optimum Temperature

They were determined by the same enzyme activity assay described aboveexcept using triolein emulsion as the substrate and that the reactiontemperature varied in the range from 30° to 100° C. The relationshipbetween the reaction temperature and the relative activity was shown inFIG. 3. The active temperature determined in the range of from 30° to100° C. was from 30° to 100° C. and the optimum temperature was from 85°to 100° C.

The active temperature and the optimum temperature were determined bythe same enzyme activity assay described above except using trioleinemulsion as the substrate in the presence of 5 mMethylenediaminetetraacetic acid (EDTA) and that the reaction temperaturevaried in the range of from 30° to 100° C. The relationship between thereaction temperature and the relative activity was shown in FIG. 5. Theactive temperature determined in the range of from 30° to 100° C. wasfrom 30° to 100° C. and the optimum temperature was from 80° to 90° C.

(6) Temperature Stability

The residual activity after treatment at pH 7 and at varying temperaturein the range of from 30° to 100° C. for 1 hour was determined by theenzyme activity assay described above. The relationship between thetreatment temperature and the residual activity was shown in FIG. 4, andthe residual activity after treatment at 80° C. was 100%.

(7) Molecular Weight

The molecular weight obtained by SDS-polyacrylamide gel electrophoresisis 32,000±2,000.

(8) Isoelectric Point

The isoelectric point obtained by SDS-polyacrylamide gel electrophoresisis 8.8±0.5.

BEST MODE FOR CARRYING OUT THE INVENTION

Now, the following examples are given to describe the present invention,but are not to be construed to limit the scope of the invention. The %in the following examples is based on weight unless otherwise indicated.

EXAMPLE 1

Cultivation of Lipase Producing Bacterium (SD709)

The liquid medium (2 ml) containing soy bean powder (2%), glucose (1%),diammonium hydrogen phosphate (0.1%), dipotassium hydrogen phosphate(0.5%), magnesium sulfate heptahydrate (0.1%) and sodium carbonate(0.3%) was put into a 18 mm diameter test tube and sterilized byautoclave at 121° C. for 20 minutes. It was inoculated with a loopful ofPseudomonas solanacearum SD709 and cultivated at 30° C. for 24 hours at130 rpm. Thereafter, the bacterial cells were separated bycentrifugation to give lipase solution. The lipase activity of thesolution was 5 U/ml.

EXAMPLE 2

Cultivation of Lipase Producing Bacterium (SD709) and Recovery of Lipase

The liquid medium (2 liter) containing soy bean powder (2%), diammoniumhydrogen phosphate (0.1%), dipotassium hydrogen phosphate (0.5%),magnesium sulfate heptahydrate (0.1%), sodium carbonate (0.3%) and Tween85 (1%) was placed on a 5 liter cultivation tub, and sterilized byautoclave at 121° C. for 20 minutes. It was inoculated with Pseudomonassolanacearum SD709 and cultivated at 30° C. for 24 hours at 1,000 rpmwith aeration and stirring. Thereafter, the bacterial cells wereseparated by centrifugation to give lipase solution. The lipase activityof the solution was 20 U/ml.

From the lipase solution obtained above the precipitate of 20% to 30%fraction was obtained by ammonium sulfate precipitation. The precipitatewas desalted by a conventional method, and freeze-dried to give lipasecrude powder.

EXAMPLE 3

Purification of Lipase

The lipase crude powder obtained in Example 2 was dissolved in 10 mMtris/hydrochloric acid buffer (pH 7), and dialyzed against 10 mMtris/hydrochloric acid buffer (pH 7) containing 10% saturated ammoniumsulfate, then treated by hydrophobic chromatography with Butyl-Toyopearl650M (trade name, Tosoh Corporation) to give the active fraction. Theactive fraction was dialyzed against 10 mM tris/hydrochloric acid buffer(pH 8) containing 0.3 mM calcium chloride dihydrate and adsorbed toDEAE-Cellulofine A-800 (trade name, Seikagaku Corporation), ion-exchangechromatography resin, equilibrated in the same buffer, and eluted withsodium chloride solution to give the active fraction. The fraction wasdesalted and freeze-dried to give the purified enzyme.

The simplicity of the freeze-dried product was confirmed bypolyacrylamide gel electrophoresis.

EXAMPLE 4:

Activity at High Temperatures in the Presence of EDTA

The activity of the lipase crude powder obtained in Example 2 wasdetermined in the presence and absence of ethylenediaminetetraaceticacid (EDTA) using triolein emulsion as the substrate at varyingtemperature in the range from 30° to 100° C. The determination in theabsence of EDTA is carried out by the same enzyme activity assaydescribed above except the reaction temperature varied in the range from30° to 100° C. And the determination in the presence of EDTA was carriedout by the same way as that in the absence of EDTA except adding EDTA.The relationship between the relative activity comparing with 100 of theactivity at 80° C. in the absence of EDTA and the reaction temperatureis shown in FIG. 5. According to FIG. 5, the active temperature of thepresent lipase is from 30° to 100° C. and the optimum temperature of itis from 80° to 90° C. even in the presence of EDTA (in the absence ofcalcium ion), and it was observed that the lipase has sufficientactivity at a high temperature.

INDUSTRIAL APPLICABILITY

The lipase of the present invention has a high thermostability, andtherefore the activity can be exhibited in hydrolysis of lipids at ahigh temperatures, and the lipase can be widely used in the fields suchas medicines, food-processing, cosmetic preparation, detergentcompounding, treatment of waste water, decomposition of fats and oils,pitch control.

Pseudomonas solanacearum SD709 (FERM P-14786) of the present inventionor its mycologically equivalent strain, or their mutants are useful forpreparation of the present invention which efficiently produces thelipase.

We claim:
 1. A purified lipase isolated from a strain of Pseudomonas,having:(a) a temperature optimum of from 80° to 90° C., determined withtriolein emulsion as a substrate in the presence of 5 mM EDTA; (b)enzymatic activity in a pH range of about 4-12; and (c) a pH optimum at6.5-9.5, determined using triolein emulsion as a substrate.
 2. Thelipase of claim 1, further having a molecular weight of 32,000±2,000Daltons, determined by SDS-polyacrylamide gel electrophoresis.
 3. Thelipase of claim 1, wherein the Pseudomonas strain is Pseudomonassolanacearum.
 4. The lipase of claim 3, wherein the Pseudomonas strainis Pseudomonas solanacearum SD709 (FERM BP-5358).
 5. A process forproducing the lipase of claim 1 by cultivating a Pseudomonassolanacearum SD709 bacterium, and recovering the lipase from the culturemedium.